Arenaviruses such as Lassa fever virus (LASV) can cause acute viral hemorrhagic fever disease in humans, to which there is no vaccine or effective treatment. Studies on basic biology, virulent mechanism, and pathogenesis of LASV are significantly hindered, partly due to the strict requirement for BSL-4 containment to work with this select agent and the lack of infectious clones. A small animal model for Lassa fever - guinea pig infected with a non-pathogenic Pichinde virus (PICV) within the same Arenaviridae family - has shown many similarities in the clinical and histopathological findings to lethal human Lassa fever infections. We have recently developed the infectious clones for two closely related PICV strains that show opposing disease outcomes in guinea pigs. In addition, we have developed safe and convenient systems to characterize arenavirus envelope glycoprotein (GPC)-mediated cell entry and LASV viral RNA synthesis in vitro. Based on our preliminary results, we hypothesize that the GPC-mediated cell entry and the polymerase protein L- dependent viral RNA synthesis represent two major virulence mechanisms of arenavirus. We propose to utilize the many molecular, genetic, and animal systems that are available in our laboratory to characterize the molecular determinants of virulent arenavirus infection in the infected hosts and to explore the virulence mechanisms of the GPC and L proteins at molecular level. Specifically, we wish to (1) characterize the molecular determinants for virulent infection in a guinea pig model of arenavirus hemorrhagic fever, (2) characterize the molecular mechanism of arenavirus GPC protein in cell entry pathways, (3) characterize the molecular mechanism of LASV L protein in viral RNA synthesis, viral replication, and virulence. These studies may lead to identification of potential therapeutic targets against Lassa fever and other arenavirus hemorrhagic fever diseases.